Project Summary/Abstract There are fundamental gaps in our knowledge about how bacterial small, noncoding RNAs (sRNAs) find and base-pair with a target mRNA and how this pairing leads to changes in gene expression. The long- term goal of this research is to elucidate the mechanisms of post-transcriptional regulation of gene expression in bacteria. The overall objective of this proposal is to understand how PNPase controls RNA stability and decay. Our central hypothesis is that PNPase plays a key role in gene regulation by protecting Hfq-bound sRNAs from degradation, degrading unbound sRNAs, and targeting paired sRNAs and mRNAs to the RNA degradosome for degradation. The experiments described herein will test this hypothesis and further define the molecular mechanisms by which PNPase binds, protects, and degrades RNAs using E. coli PNPase as the model system. The significance of the proposed research is that it will advance our knowledge of a novel activity of PNPase, stabilizing RNAs, and increase our understanding of sRNA-mediated regulation of gene expression, which is integral to bacterial stress responses and include antibiotic resistance mechanisms. The research proposed in this application is innovative, because it will challenge the existing paradigm that describes PNPase solely as an RNA degrading enzyme that only recognizes the 3' ends of RNAs. In Aim 1, we will define the molecular mechanism of PNPase-mediated RNA protection. Our working hypothesis is that PNPase protects Hfq-bound sRNAs from degradation by other RNases by occluding potential binding or cleavage sites. Using genetic, molecular, and biochemical approaches we will define the sites of interactions between PNPase and sRNAs, test the role of the exoribonuclease activity of PNPase in sRNA protection, and assess the contribution of other RNases to sRNA decay in the absence of PNPase. In Aim 2, the mechanism by which PNPase mediates decay of sRNAs and target mRNAs will be investigated. Our working hypothesis is that PNPase degrades sRNAs not bound to Hfq, and targets certain paired sRNAs and mRNAs to the RNA degradosome for degradation. The RNA degradosome is comprised of the endoribonuclease RNase E, glycolytic enzyme enolase, the RNA helicase RhlB, and PNPase. Through a comprehensive set of genetic, molecular, and biochemical approaches, we will define the substrate specificity of PNPase and the role of particular residues in the degradation of RNAs. Finally, we will define the sites of RNase E that interact with sRNAs and test whether or not PNPase contributes to recruitment of RNAs to RNase E upon sRNA-mRNA pairing. Since PNPase is highly conserved among bacteria and eukaryotes, understanding the molecular mechanism of how PNPase controls RNA stability in E. coli will provide insight into RNA metabolism in most living organisms.